Outline

Background: Detection of disseminated tumor cells (DTC) in bone marrow (BM) is routinely performed with cytokeratin (CK) antibodies. The detection rate is influenced by false-positive staining and CK expression of bone marrow cells. We therefore used preanalytical immunomagnetic tumor cell enrichment followed by molecular detection of CK19 transcripts for the verification of bright-field immunocytology results.

Material and Methods: Mononuclear cells from bone marrow of 135 patients with primary breast cancer were used for standardized cytospin immunocytology. Tumor cells were detected by 5D3 staining using Ventana enhanced FastRed. Parallel DTC consists of immunomagnetic tumor cell selection using the cell membrane glycoproteins EpCAM and MUC-1 as targets. The breast carcinoma-associated transcripts EpCAM (EP), MUC-1 (MU) and HER-2 (HE) were analysed by multiplex RT-PCR. CK 19 transcripts were determined by nested RT-PCR. Sensitivity for every single transcript was adjusted to 1 tumor cells in 1x107 BM cells.

Results: Using acetic acid and periodate blocking steps before immunocytology 3 from 135 patients were scored positive (2, 6 and 8 tumor cells/106 BM cells) upon computer assisted automated picture analysis (Chromavision ACIS). Multiplex PCR results revealed EpCAM and MUC-1 positivity in 84 and 81% of the BM probes, which corresponds well with immunocytological results otained with VU1D9 (EpCAM) and BM2 (MUC-1) staining. Both antigens are expressed by normal BM cells of the erythroid lineage. However, tumor-associated CK19 transcripts were detected in only 5 of 135 patients. Concordant results of cytology and CK19 RT-PCR were found 132 BM samples, with two positive and 130 negative probes.

Discussion: Tumor cells in bone marrow of patients with primary breast cancer are routinely analysed by bright-field cytology using CK specific antibodies A45B/B3 (CK7/8/18,19), AE1/AE3 (subfamilies A and B) and 5D3 (CK8/18) with the APAAP technique. We have shown that rare stroma cells of normal bone marrow are positive for various antigens including CK polypeptides 7, 8, 18 and 19. Elimination of these cells by size exclusion results in a detection rate of 2,6%. These low detection rates could be verified by preanalytical immunomagnetic selection followed by CK19 RT-PCR. We conclude that immunocytological CK analysis in bone marrow is dramatically impaired by staining of normal bone marrow stroma cells.